In 2010, the Benner group announced the development of four innovations relevant to tools to detect human immunodeficiency virus (HIV) in complex biological samples: (a) An artificially expanded genetic information systems (AEGIS) that supports six nucleotide PCR, allowing independent amplification of small amounts of HIV RNA without interference from other DNA in the environment. (b) A self-avoiding molecular recognition system (SAMRS) that supports essentially unlimited multiplexing in DNA probing, priming, and multiplexed PCR amplification. (c) Procedures that convert standard DNA into AEGIS-containing DNA, supporting downstream orthogonal capture that allows DNA-targeted assays to be flexible and adaptive, possibly allowing new targets to be added to a multiplexed assay kit without demanding a reworking of the parts of that kit already targeted. (d) Reversible terminators that, as triphosphates, are hypothesized to allow detection and relative quantitation of variant HIV sequences. We hypothesize that by combining these innovations, we can improve HIV diagnostics tools, expanding their power to detect fewer virions in more complex biological environments with greater dynamic range and greater subtype specificity, together greater multiplexing. Further, these technologies should deliver flexibility; it should be possible to rapidly add capabilities to detect new variants, co-incident infectious agents, or even identify previously unknown variants at specific sites in the HIV genome in the course of diagnosing HIV infections. To test this hypothesis, we will perform a staged series of assay development, adding each of these innovations in series to increasingly challenging problems in the detection of HIV target sequences, starting with singleplexed detection of single HIV targets in relatively simple environments, adding innovations as we lower the amount of target molecules, increase the level of multiplexing, and make the environment more complex. At each stage, we will drive the system to fail, and note the parameters (sensitivity, complexity, multiplexing level) at which the system fails. These define a parameter space which provides a metric for progress. This project will also make available as deliverables kits of primers, probes, and detection capture beads, to be provided HIV researchers interested in benchmarking or using them. Although technology from the Benner laboratory stands behind the branched DNA (bDNA) 3.0 tool now widely used to measure HIV viral load, this is the first time that the Benner laboratory has sought funding for AIDS research. Thus, a further goal of this work will be to allow innovations from the Benner laboratory to be more widely used to solve the many HIV-related problems at the NIAID. This will help the NIAID help meet the goal established by the National HIV/AIDS Strategy of increasing the awareness of HIV status from 79% to 90% by 2015 in the US.